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• W2075519389 abstract "The polycystic kidney (PCK) rat is an animal model of Caroli's disease with congenital hepatic fibrosis, in which the mechanism of progressive hepatic fibrosis remains unknown. This study aimed to clarify the mechanism of hepatic fibrosis of the PCK rat from the viewpoint of the contribution of pathological cholangiocytes. In liver sections of the PCK rats, intrahepatic bile ducts were constituted by two different phenotypes: bile ducts lined by cuboidal-shaped and flat-shaped cholangiocytes. The flat-shaped cholangiocytes showed reduced immunohistochemical expression of the biliary epithelial marker cytokeratin 19 and positive immunoreactivity for vimentin and fibronectin. When cultured cholangiocytes of the PCK rat were treated with transforming growth factor (TGF)-β1, a potent inducer of epithelial-mesenchymal transition, induction of vimentin, fibronectin, and collagen expression occurred in the PCK cholangiocytes. Although the TGF-β1 treatment reduced cytokeratin 19 expression, the epithelial cell features characterized by the expression of E-cadherin and zonula occludens-1 was maintained, and α-smooth muscle actin expression was not induced in the cholangiocytes. Cholangiocytes of the PCK rat may acquire mesenchymal features in response to TGF-β1 and participate in progressive hepatic fibrosis by producing extracellular matrix molecules, which seems to be a different event from epithelial-mesenchymal transition. The polycystic kidney (PCK) rat is an animal model of Caroli's disease with congenital hepatic fibrosis, in which the mechanism of progressive hepatic fibrosis remains unknown. This study aimed to clarify the mechanism of hepatic fibrosis of the PCK rat from the viewpoint of the contribution of pathological cholangiocytes. In liver sections of the PCK rats, intrahepatic bile ducts were constituted by two different phenotypes: bile ducts lined by cuboidal-shaped and flat-shaped cholangiocytes. The flat-shaped cholangiocytes showed reduced immunohistochemical expression of the biliary epithelial marker cytokeratin 19 and positive immunoreactivity for vimentin and fibronectin. When cultured cholangiocytes of the PCK rat were treated with transforming growth factor (TGF)-β1, a potent inducer of epithelial-mesenchymal transition, induction of vimentin, fibronectin, and collagen expression occurred in the PCK cholangiocytes. Although the TGF-β1 treatment reduced cytokeratin 19 expression, the epithelial cell features characterized by the expression of E-cadherin and zonula occludens-1 was maintained, and α-smooth muscle actin expression was not induced in the cholangiocytes. Cholangiocytes of the PCK rat may acquire mesenchymal features in response to TGF-β1 and participate in progressive hepatic fibrosis by producing extracellular matrix molecules, which seems to be a different event from epithelial-mesenchymal transition. The polycystic kidney (PCK) rat is an established animal model of Caroli's disease with congenital hepatic fibrosis (CHF), as well as a slowly progressive model of autosomal recessive polycystic kidney disease (ARPKD).1Sanzen T Harada K Yasoshima M Kawamura Y Ishibashi M Nakanuma Y Polycystic kidney rat is a novel animal model of Caroli's disease associated with congenital hepatic fibrosis.Am J Pathol. 2001; 158: 1605-1612Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar Caroli's disease with CHF is a hepatic manifestation of ARPKD, and the hepatic lesions of ARPKD are characterized by multiple segmental and saccular dilation of the intrahepatic bile ducts and progressive and unresolving portal fibrosis.2Nakanuma Y Terada T Ohta G Kurachi M Matsubara F Caroli's disease in congenital hepatic fibrosis and infantile polycystic disease.Liver. 1982; 2: 346-354Crossref PubMed Scopus (77) Google Scholar, 3Torres VE Harris PC Mechanisms of disease: autosomal dominant and recessive polycystic kidney diseases.Nat Clin Pract Nephrol. 2006; 2: 40-55Crossref PubMed Scopus (237) Google Scholar, 4Sweeney Jr, WE Avner ED Molecular and cellular pathophysiology of autosomal recessive polycystic kidney disease (ARPKD).Cell Tissue Res. 2006; 326: 671-685Crossref PubMed Scopus (84) Google Scholar Persistence or lack of remodeling of the embryonic ductal plate is regarded as an essential precursor of the hepatic lesions of Caroli's disease.5Desmet VJ Congenital diseases of intrahepatic bile ducts: variations on the theme “ductal plate malformation”.Hepatology. 1992; 16: 1069-1083Crossref PubMed Scopus (475) Google Scholar Symptoms from the liver disease often result from complications of dilated ducts or hepatic fibrosis, and the spontaneous course of Caroli's disease with CHF is dominated by biliary infection such as recurrent ascending cholangitis and sepsis.2Nakanuma Y Terada T Ohta G Kurachi M Matsubara F Caroli's disease in congenital hepatic fibrosis and infantile polycystic disease.Liver. 1982; 2: 346-354Crossref PubMed Scopus (77) Google Scholar, 3Torres VE Harris PC Mechanisms of disease: autosomal dominant and recessive polycystic kidney diseases.Nat Clin Pract Nephrol. 2006; 2: 40-55Crossref PubMed Scopus (237) Google Scholar, 4Sweeney Jr, WE Avner ED Molecular and cellular pathophysiology of autosomal recessive polycystic kidney disease (ARPKD).Cell Tissue Res. 2006; 326: 671-685Crossref PubMed Scopus (84) Google ScholarMutations to orthologous genes, PKHD1/Pkhd1, have been identified in ARPKD patients and the PCK rat.6Ward CJ Hogan MC Rossetti S Walker D Sneddon T Wang X Kubly V Cunningham JM Bacallao R Ishibashi M Milliner DS Torres VE Harris PC The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein.Nat Genet. 2002; 30: 259-269Crossref PubMed Scopus (580) Google Scholar, 7Onuchic LF Furu L Nagasawa Y Hou X Eggermann T Ren Z Bergmann C Senderek J Esquivel E Zeltner R Rudnik-Schoneborn S Mrug M Sweeney W Avner ED Zerres K Guay-Woodford LM Somlo S Germino GG PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats.Am J Hum Genet. 2002; 70: 1305-1317Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar Developing and mature intrahepatic bile ducts express the PKHD1 protein, fibrocystin, whereas bile ducts of ARPKD patients lack its expression.8Ward CJ Yuan D Masyuk TV Wang X Punyashthiti R Whelan S Bacallao R Torra R LaRusso NF Torres VE Harris PC Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia.Hum Mol Genet. 2003; 12: 2703-2710Crossref PubMed Scopus (260) Google Scholar Mice with targeted mutation of Pkhd1 develop cystic biliary dysgenesis and portal fibrosis.9Moser M Matthiesen S Kirfel J Schorle H Bergmann C Senderek J Rudnik-Schoneborn S Zerres K Buettner R A mouse model for cystic biliary dysgenesis in autosomal recessive polycystic kidney disease (ARPKD).Hepatology. 2005; 41: 1113-1121Crossref PubMed Scopus (72) Google Scholar In the PCK rat, cholangiocytes possess short and malformed cilia that do not express fibrocystin.10Masyuk TV Huang BQ Ward CJ Masyuk AI Yuan D Splinter PL Punyashthiti R Ritman EL Torres VE Harris PC LaRusso NF Defects in cholangiocyte fibrocystin expression and ciliary structure in the PCK rat.Gastroenterology. 2003; 125: 1303-1310Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 11Masyuk TV Huang BQ Masyuk AI Ritman EL Torres VE Wang X Harris PC Larusso NF Biliary dysgenesis in the PCK rat, an orthologous model of autosomal recessive polycystic kidney disease.Am J Pathol. 2004; 165: 1719-1730Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 12Muff MA Masyuk TV Stroope AJ Huang BQ Splinter PL Lee SO Larusso NF Development and characterization of a cholangiocyte cell line from the PCK rat, an animal model of autosomal recessive polycystic kidney disease.Lab Invest. 2006; 86: 940-950Crossref PubMed Scopus (33) Google Scholar Despite the identification of the genetic defect of the disease, the pathophysiology of the bile duct dilation and hepatic fibrosis are not fully understood. With regard to the mechanism of progressive dilation of intrahepatic bile ducts, our pervious studies have shown that activation of the signaling pathway mediated by epidermal growth factor receptor is involved in the abnormal cholangiocyte growth in the PCK rat.13Sato Y Harada K Kizawa K Sanzen T Furubo S Yasoshima M Ozaki S Ishibashi M Nakanuma Y Activation of the MEK5/ERK5 cascade is responsible for biliary dysgenesis in a rat model of Caroli's disease.Am J Pathol. 2005; 166: 49-60Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 14Sato Y Harada K Furubo S Kizawa K Sanzen T Yasoshima M Ozaki S Isse K Sasaki M Nakanuma Y Inhibition of intrahepatic bile duct dilation of the polycystic kidney rat with a novel tyrosine kinase inhibitor gefitinib.Am J Pathol. 2006; 169: 1238-1250Abstract Full Text Full Text PDF PubMed Scopus (22) Google ScholarIn most types of chronic liver diseases, activated hepatic stellate cells/myofibroblasts play major roles in hepatic fibrosis by producing extracellular matrix molecules.15Bataller R Brenner DA Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Scopus (4019) Google Scholar These fibrogenic processes usually occur after hepatocellular damage by various causative agents, whereas lack of necroinflammatory change in hepatic parenchyma is a histological feature of Caroli's disease with CHF.2Nakanuma Y Terada T Ohta G Kurachi M Matsubara F Caroli's disease in congenital hepatic fibrosis and infantile polycystic disease.Liver. 1982; 2: 346-354Crossref PubMed Scopus (77) Google Scholar In fact, our previous study demonstrated that α-smooth muscle actin (α-SMA)-expressing myofibroblasts were negligible in hepatic parenchyma in liver sections of patients with Caroli's disease with CHF.16Ozaki S Sato Y Yasoshima M Harada K Nakanuma Y Diffuse expression of heparan sulfate proteoglycan and connective tissue growth factor in fibrous septa with many mast cells relate to unresolving hepatic fibrosis of congenital hepatic fibrosis.Liver Int. 2005; 25: 817-828Crossref PubMed Scopus (28) Google Scholar Therefore, hepatic fibrosis in Caroli's disease with CHF seems to be mediated by other cell types than hepatic stellate cells.Recently, many studies have demonstrated that epithelial cells have an ability to acquire mesenchymal features, providing proof of principle for the process of epithelial-mesenchymal transition (EMT).17Kang Y Massague J Epithelial-mesenchymal transitions: twist in development and metastasis.Cell. 2004; 118: 277-279Abstract Full Text Full Text PDF PubMed Scopus (1227) Google Scholar, 18Zavadil J Bottinger EP TGF-beta and epithelial-to-mesenchymal transitions.Oncogene. 2005; 24: 5764-5774Crossref PubMed Scopus (1368) Google Scholar EMT has been implicated in a variety of biological processes such as fibrogenesis as well as embryonic development and tumor progression. For example, renal tubular epithelial cells undergo EMT that is linked to the pathogenesis of renal interstitial fibrosis.19Liu Y Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention.J Am Soc Nephrol. 2004; 15: 1-12Crossref PubMed Scopus (950) Google Scholar In idiopathic pulmonary fibrosis, alveolar epithelial cells can serve as a source of myofibroblasts through EMT.20Willis BC Liebler JM Luby-Phelps K Nicholson AG Crandall ED du Bois RM Borok Z Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis.Am J Pathol. 2005; 166: 1321-1332Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar Transforming growth factor (TGF)-β is known to be the most potent inducer of EMT, and it initiates morphological transition of the cells from an epithelial to a fibroblastic appearance, accompanied by a loss of epithelial cell markers such as E-cadherin and a gain of mesenchymal cell markers such as vimentin, fibronectin, and N-cadherin.18Zavadil J Bottinger EP TGF-beta and epithelial-to-mesenchymal transitions.Oncogene. 2005; 24: 5764-5774Crossref PubMed Scopus (1368) Google Scholar, 19Liu Y Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention.J Am Soc Nephrol. 2004; 15: 1-12Crossref PubMed Scopus (950) Google ScholarAccording to our previous study,14Sato Y Harada K Furubo S Kizawa K Sanzen T Yasoshima M Ozaki S Isse K Sasaki M Nakanuma Y Inhibition of intrahepatic bile duct dilation of the polycystic kidney rat with a novel tyrosine kinase inhibitor gefitinib.Am J Pathol. 2006; 169: 1238-1250Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar administration of a tyrosine kinase inhibitor inhibited intrahepatic bile duct dilation of the PCK rat, and this inhibition resulted in an improvement of hepatic fibrosis. In a rat model of biliary fibrosis, bile duct epithelial cells participate in the fibrotic process by producing connective tissue growth factor.21Sedlaczek N Jia JD Bauer M Herbst H Ruehl M Hahn EG Schuppan D Proliferating bile duct epithelial cells are a major source of connective tissue growth factor in rat biliary fibrosis.Am J Pathol. 2001; 158: 1239-1244Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar In addition, a recent study has demonstrated that bile duct epithelial cells can undergo EMT, thereby contributing to hepatic fibrosis in a mouse model of bile duct ligation.22Xia JL Dai C Michalopoulos GK Liu Y Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation.Am J Pathol. 2006; 168: 1500-1512Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar These observations suggest that cholangiocytes contribute to hepatic fibrosis under several pathophysiological conditions, and the fibrocystic liver disease may be a candidate of such diseases. Using the PCK rat as an animal model of Caroli's disease with CHF, this study was performed to clarify the mechanism of hepatic fibrosis of the PCK rat from the view point of the contribution of pathological cholangiocytes.Materials and MethodsAnimals and TissuesPCK rats were maintained at the Laboratory Animal Institute of Kanazawa University Graduate School of Medicine. Normal (Crj:CD) rats were purchased from Charles River Japan (Sagamihara, Japan). Livers were removed from fetal (21 days of gestation), neonatal, and adult (1 day, 3 weeks, 2 months, 6 months, and 10 months old) rats. They were immersed in 10% formalin neutral buffer solution (pH 7.4) and embedded in paraffin. More than 10 serial sections, 4 μm thick, were cut from each paraffin block. Several of these sections were stained with hematoxylin and eosin (H&E) and Azan-Mallory, and the remainder was subjected to the immunohistochemical analysis. Parts of the tissue were immediately frozen in liquid nitrogen for use in the reverse transcriptase-polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay (ELISA). All animal studies were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at Takara-machi Campus of Kanazawa University, Kanazawa, Japan.Cell Culture of PCK CholangiocytesBiliary epithelial cells were isolated, purified, and cultured from the intrahepatic large bile ducts of 8-week-old male PCK rats and normal rats as described previously.13Sato Y Harada K Kizawa K Sanzen T Furubo S Yasoshima M Ozaki S Ishibashi M Nakanuma Y Activation of the MEK5/ERK5 cascade is responsible for biliary dysgenesis in a rat model of Caroli's disease.Am J Pathol. 2005; 166: 49-60Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar The 8th to 12th subcultured cholangiocytes were used for the study. Cells were set on type I collagen-coated cell culture dishes (Iwaki, Scitech Div., Chiba, Japan) covered with a standard culture medium composed of Dulbecco's modified Eagle's medium/F-12 (Life Technologies, Inc., Rockville, MD), 10% bovine growth serum (HyClone, Logan, UT), 1% ITS+ (Becton Dickinson, Bedford, MA), 5 μmol/L forskolin (Wako Pure Chemical, Osaka, Japan), 12.5 mg/ml of bovine pituitary extract (Kurabo Industries, Osaka, Japan), 1 μmol/L dexamethasone (Sigma, St. Louis, MO), 5 μmol/L triiodo-thyronine (Sigma), 5 mg/ml of glucose (Sigma), 25 mmol/L sodium bicarbonate (Sigma), 1% antibiotics-antimycotic (Life Technologies, Inc.), and 20 ng/ml of epidermal growth factor (Upstate Biotechnology, Lake Placid, NY) at 37°C in an atmosphere of 5% CO2. The standard culture medium was changed every 2 days until subconfluent.Treatment of PCK Cholangiocytes with TGF-β1At subconfluent state on type I collagen-coated cell culture dishes, cholangiocytes of PCK and normal rats were incubated with the standard medium or that containing TGF-β1 (2 ng/ml; R&D Systems, Inc., Minneapolis, MN) for 3 and 7 days. The culture medium was changed every day. After the treatment, the cultured cells and their supernatant were stored for the following experiments. To determine further the effects of direct cell contact with the basement membrane components, PCK cholangiocytes were cultured on type IV collagen- or laminin-coated cell culture dishes (BD Biosciences, Bedford, MA), and similarly treated with TGF-β1.RT-PCRTotal RNA (1 μg) was extracted from the liver and cultured cholangiocytes using an RNA extraction kit (RNeasy mini; Qiagen, Tokyo, Japan) and was used to synthesize cDNA with reverse transcriptase (ReverTra Ace; Toyobo Co., Osaka, Japan). The sequences of the primers and conditions for PCR used are shown in Table 1. PCR amplification was performed in a reaction mixture containing 0.2 mmol/L dNTPs, 1 μmol/L each 5′- and 3′-primers, and 2.5 U of TaqDNA polymerase (Takara EX Taq; Takara Bio, Shiga, Japan). For each reaction, an initial denaturation cycle of 94°C for 3 minutes and a final cycle of 72°C for 10 minutes were incorporated. The PCR products were subjected to 2% agarose gel electrophoresis and stained with ethidium bromide.Table 1Sequences of the Primers and PCR Conditions Used in This StudyGeneSequencesAnnealing temperature (°C)PCR cyclesCK195′-GTACCAGAAGCAGGGACCCG-3′60225′-TTCCAGGGCAGCTTTCATGC-3′E-cadherin5′-GAGGGTTTTCACTGGTTGTT-3′55285′-GTAAAGACACTCTGGAGGTG-3′Vimentin5′-TTCAAGAACACCCGCACCAAC-3′55295′-GCCTTCCAGCAGCTTCCTGTA-3′Procollagen type I5′-CATAAAGGGTCATCGTGGCTTC-3′65355′-GTGATAGGTGATGTTCTGGGAG-3′Fibronectin5′-CCTACAACATCATAGTGGAG-3′55355′-CTTGTAGTTGACACCGTTGT-3′α-SMA5′-CATCCACGAAACCACCTATA-3′55405′-CTGTTTGCTGATCCACATCT-3′TGF-β15′-CAATTCCTGGCGTTACCTTG-3′60355′-GAAGCAGTAGTTGGTATCCA-3′TβR-II5′-GTGAAGAACGATTTGACCTG-3′50405′-GTTGTCTTTCATGCTCTCCA-3′β-Actin5′-ACCTTCAACACCCCAGCCATGTACG-3′60255′-CTGATCCACATCTGCTGGAAGGTGG-3′CK, cytokeratin; SMA, smooth muscle actin; TGF, transforming growth factor; TβR-II, TGF-β type II receptor. Open table in a new tab Immunofluorescence Confocal MicroscopyCholangiocytes of PCK and normal rats grown on type I collagen-coated coverslips were treated with TGF-β1 (2 ng/ml) for 3 and 7 days. Then they were fixed with 4% paraformaldehyde for 15 minutes and permeabilized for 3 minutes with 0.1% Triton X-100. After blocking, the cells were incubated for 1 hour at room temperature with primary antibodies against pan-cytokeratin (CK) (1:600; DakoCytomation, Glostrup, Denmark), zonula occludens-1 (ZO-1) (1:200; Zymed, South San Francisco, CA), and vimentin (1:600, DakoCytomation). Alexa-488 and Alexa-568 (10 μg/ml; Molecular Probes, Eugene, OR) were used as a secondary antibody, and nuclei were stained with 4′,6-diamidino-2-phenylindole.Western Blot AnalysisProteins were extracted from cultured cholangiocytes using T-PER tissue protein extraction reagent (Pierce Chemical Co., Rockford, IL), and the total protein was measured spectrometrically. First, 50 μg of the protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide electrophoresis and then electrophoretically transferred on to a nitrocellulose membrane. The membrane was incubated with primary antibodies against CK19 (1:100; Novocastra, Newcastle on Tyne, UK), E-cadherin (1:100, Zymed), and actin (1:3000; Abcam Inc., Cambridge, MA). The protein expression was detected using an EnVison+ system (DakoCytomation). 3,3′-Diaminobenzidine tetrahydrochloride was used as the chromogen. Semiquantitative analysis of the results was performed using the public domain NIH image software (National Institutes of Health, Bethesda, MD). The fold difference compared with actin expression was calculated.ELISAConcentrations of TGF-β1 were determined using an ELISA kit (R&D Systems) according to the manufacturer's instructions. Briefly, serum and protein extracts from the liver of the PCK and normal rats were added to a 96-well plate coated with a monoclonal antibody for TGF-β1 and incubated for 2 hours at room temperature. After washing, an enzyme-linked polyclonal antibody for TGF-β1 was added and incubated for 2 hours. Color development was performed using a substrate solution, and its absorbance at 450 nm was measured. Total protein was measured spectrometrically, and TGF-β1/total protein concentration was calculated for each sample.For the determination of concentration of fibronectin, an ELISA kit (Biomedical Technologies Inc, Stoughton, MA) was used. Briefly, cell culture supernatant was incubated with a specific primary antiserum against fibronectin in a 96-well plate for 1 hour at 37°C. For negative control wells, the standard culture medium was added. A second incubation was performed with an alkaline phosphatase-fibronectin conjugate. Separation of the antibody-bound and -free fractions was achieved by a second antibody that was precoated to the wells. After incubation with substrate, the amount of bound enzyme was determined by absorption at 405 nm.Measurement of Collagen ContentThe collagen content in cell culture supernatant was measured using the Sircol collagen assay kit (Biocolor Ltd., Belfast, UK) according to the manufacturer's instructions. Briefly, Sirius red reagent (50 μl) was added to each culture supernatant (50 μl) and mixed for 30 minutes. The collagen-dye complex was precipitated by centrifugation at 15,000 × g for 5 minutes, washed with ethanol, and dissolved in 0.5 mol/L sodium hydroxide. Finally, the samples were introduced into a microplate reader, and the absorbance was determined at 540 nm. Because the standard culture medium contained detectable amounts of collagen, the data were expressed as an increase in collagen content by subtracting the measured value for the standard culture medium.ImmunohistochemistryImmunostaining was performed for formalin-fixed, paraffin-embedded liver sections. After deparaffinization and blocking of the endogenous peroxidase, the sections were incubated overnight at 4°C with individual primary antibodies listed in Table 2. Then, the sections were incubated with secondary antibody conjugated to the peroxidase-labeled polymer, EnVison+ system (DakoCytomation). Color development was performed using 3,3′-diaminobenzidine tetrahydrochloride, and the sections were counterstained with hematoxylin. Control sections were evaluated by substitution of the primary antibodies with corresponding nonimmunized serum, which resulted in no signal detection.Table 2Primary Antibodies Used for the Immunohistochemical AnalysisAntibody specificityCloneSourceDilutionPan-CKPolyclonalDakoCytomation, Glostrup, Denmark1:600*), by incubating with 1 mg/ml trypsin for 15 minutes at 37°C, (CK7OV-TL 12/30DakoCytomation1:50*), by incubating with 1 mg/ml trypsin for 15 minutes at 37°C, (CK19b170Novocastra, Newcastle upon Tyne, UK1:100†), and by incubating 20 mg/ml of proteinase K for 6 minutes at room temperature (E-cadherin4A2C7Zymed, South San Francisco, CA1:200*), by incubating with 1 mg/ml trypsin for 15 minutes at 37°C, (VimentinV9DakoCytomation1:600*), by incubating with 1 mg/ml trypsin for 15 minutes at 37°C, (DesminD33DakoCytomation1:200α-SMA1A4DakoCytomation1:200N-cadherinPolyclonalCalbiochem, La Jolla, CA1:100*), by incubating with 1 mg/ml trypsin for 15 minutes at 37°C, (FibronectinPolyclonalDakoCytomation1:200‡). CK, cytokeratin; SMA, smooth muscle actin; TβR-I, transforming growth factor-β type I receptor; TβR-II, TGF-β type II receptor.TβR-IPolyclonalSanta Cruz Biotechnology, Inc., Santa Cruz, CA1:50†), and by incubating 20 mg/ml of proteinase K for 6 minutes at room temperature (TβR-IIPolyclonalSanta Cruz Biotechnology, Inc.1:50†), and by incubating 20 mg/ml of proteinase K for 6 minutes at room temperature (Phospho-Smad2PolyclonalCell Signaling Technology, Inc., Danvers, MA1:100‡). CK, cytokeratin; SMA, smooth muscle actin; TβR-I, transforming growth factor-β type I receptor; TβR-II, TGF-β type II receptor.Antigen retrieval was performed by microwaving in 10 mmol/L citrate buffer pH 6.0 (* ), by incubating with 1 mg/ml trypsin for 15 minutes at 37°C, († ), and by incubating 20 mg/ml of proteinase K for 6 minutes at room temperature (‡ ). CK, cytokeratin; SMA, smooth muscle actin; TβR-I, transforming growth factor-β type I receptor; TβR-II, TGF-β type II receptor. Open table in a new tab Double Immunostaining and Histological AssessmentDouble immunostaining of pan-CK and vimentin was conducted as follows. The deparaffinized liver sections were incubated overnight at 4°C with anti-pan-CK (1:200) and then incubated using the EnVision+ system (DakoCytomation). Color development was performed using the Vector Blue alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA). The detection of vimentin was performed as above. Stained liver sections were visualized under a light microscope, and the digital images were acquired and reproduced on a computer. Areas of lumen of pan-CK single-positive bile ducts, and those of pan-CK- and vimentin-double-positive bile ducts were measured using image analysis software (WinROOF version 3.6; Mitani Corp., Tokyo, Japan). The areas of interest were expressed as a percentage of the total tissue.The extent of fibrosis around bile ducts was determined using sections stained with Azan-Mallory. The digital images of the sections were reproduced on a computer, and a color threshold was applied at a level that separated periductal fibrosis from nonfibrotic tissue. Areas of periductal fibrosis and those of bile duct lumen were measured using WinROOF software (Mitani Corp.). Periductal fibrosis score was expressed as a ratio of area of periductal fibrosis/area of bile duct lumen.StatisticsThe mean ± SD was calculated for all parameters. Statistical differences were determined using the Mann-Whitney U-test or analysis of variance. A P value <0.05 was accepted as the level of statistical significance.ResultsTwo Different Types of Intrahepatic Bile Ducts of the PCK RatIn liver sections of the PCK rats stained with H&E, two different types of intrahepatic bile ducts could be recognized, ie, bile ducts lined by cuboidal-shaped cholangiocytes [Figure 1, A (arrowheads) and B] and those lined by flat-shaped cholangiocytes [Figure 1, A (arrows) and C]. The bile ducts lined by cuboidal-shaped cholangiocytes (C-type) were a predominant phenotype in the liver, showing progressive cystic dilation with increasing age. The bile ducts lined by flat-shaped cholangiocytes (F-type) were not observed under the age of 1 day but seemed to distribute more frequently with advancing age. F-type had fusiform-shaped lumen with uneven nuclear spacing and lower nuclear density than those of C-type (Figure 1C). Some F-type bile ducts had enlarged nuclei. In the elderly rats, mildly dilated F-type bile ducts were observed, but F-type seemed not to show progressive cystic dilation (Figure 1A). At the ages of 6 and 10 months, cholangitis became a frequent histological finding in C-type with accumulation of polymorphonuclear leukocytes in their lumen. By contrast, F-type never associated with suppurative cholangitis even in the elderly rats, despite the close spatial distance between C- and F-types (Figure 1D).Both C- and F-types accompanied with progressive dense fibrosis around them, and F-type occasionally showed fibrous scar-like appearance in the elderly rats (Figure 1E, arrows). Despite dense fibrotic response in the portal tracts, α-SMA-expressing portal fibroblasts were scanty at any age of the rats around C- and F-type bile ducts (Figure 1F). Also, α-SMA-expressing myofibroblasts were negligible in hepatic parenchyma of the PCK rats (Figure 1F).Frequency of Distribution of Two Different Type Bile Ducts in the PCK Liver during AgingDouble immunostaining of pan-CK (colored by the Vector Blue reaction) and vimentin (colored by the benzidine reaction) was performed for the PCK liver sections, and the frequency of distribution of C- and F-type bile ducts was determined. As demonstrated later, cholangiocytes constituting F-type bile ducts had mesenchymal features. In the analysis, pan-CK single-positive bile ducts (Figure 1G) were regarded as C-type, and bile ducts showing pan-CK and vimentin double-positive with reduced signal intensity of pan-CK (Figure 1H) were regarded as F-type. As shown in Figure 1I, the percentage of areas of C-type bile duct lumen to the whole liver tissue progressively increased with aging, and C-type was invariably predominant at any age of the rats. F-type first appeared at 3 weeks of age, and its frequency gradually increased up to 10 months of age.Progressive Fibrosis around Two Different Type Bile Ducts in the PCK Liver during AgingFibrosis around the two different type bile ducts was assessed using liver sections stained with Azan-Mallory. C- and F-type bile ducts in Azan-Mallory-stained sections were determined" @default.
• W2075519389 created "2016-06-24" @default.
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• W2075519389 date "2007-12-01" @default.
• W2075519389 modified "2023-10-17" @default.
• W2075519389 title "Cholangiocytes with Mesenchymal Features Contribute to Progressive Hepatic Fibrosis of the Polycystic Kidney Rat" @default.
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